This invention pertains generally to biological assays of components of a fatty acid synthase, and more particularly, to the use of scintillation proximity assays suitable for detecting and measuring activities such as that of xcex2-ketoacyl-acyl carrier protein synthase III.
The pathway for the biosynthesis of saturated fatty acids is very similar in prokaryotes and eukaryotes. However, while the chemical reactions may not vary, the organization of the biosynthetic apparatus is very different. Vertebrates and yeast possess type I fatty acid synthases (FASs) in which all of the enzymatic activities of the fatty acid biosynthetic pathway are encoded on one or two polypeptide chains, respectively, and an acyl carrier protein (ACP) is an integral part of the complex. In contrast, in most bacterial and plant type II FASs, each of the reactions is catalyzed by distinct monofunctional enzymes and the ACP is a discrete protein. Mycobacteria are unique in that they possess both type I and II FASs; the former is involved in basic fatty acid biosynthesis whereas the latter is involved in synthesis of complex cell envelope lipids such as mycolic acids.
The fatty acid biosynthetic process is divided into three phases: initiation, elongation and termination. The elongation phase is cyclical, in that acyl thioester (typically attached to an acyl carrier protein) is repeatedly elongated two carbons at a time using malonyl CoA as the 2-carbon xe2x80x9cextenderxe2x80x9d unit.
In type II fatty acid biosynthesis (refer to FIG. 1) the first elongation step or initiation step is the condensation of malonyl-ACP (MACP) with an acyl-CoA which is catalyzed by xcex2-ketoacyl-acyl carrier protein synthase III (KAS III or FabH, see definitions). The product of this reaction is xcex2-ketoacyl ACP which then enters the reduction component of the cycle by undergoing ketoester reduction by NADPH-dependent xcex2-ketoacyl-ACP reductase (FabG, xe2x80x9cKRxe2x80x9d in FIG. 1). Subsequent dehydration by xcex2-hydroxyacyl-ACP dehydrase (either FabA or FabZ, represented by xe2x80x9cDHxe2x80x9d in FIG. 1) leads to trans-2-enoyl-ACP which is in turn converted to acyl-ACP by NADH-dependent enoyl-ACP reductase (FabI, xe2x80x9cERxe2x80x9d in FIG. 1). Synthases KAS I and KAS II (also known as FabB and FabF, see definitions) then catalyze a 2-carbon elongation of acyl-ACP via condensation with MACP, followed by ketoester reduction, dehydration, and enoyl reduction as described above. Further rounds of this cycle, adding two carbon atoms per cycle, eventually lead to production of a fatty acid of the desired length, whereupon the cycle is stopped largely due to feedback inhibition of KASIII (Heath, et al, (1996), J.Biol.Chem. 271, 1833-1836). KASIII is therefore a major biosynthetic enzyme which is also a key regulatory point in the overall biosynthetic pathway (Heath, R. J. and Rock, C. O. 1996. J.Biol.Chem. 271, 1833-1836; Heath, R. J. and Rock, C. O. 1996. J.Biol.Chem. 271, 10996-11000). KASIII from different organisms exhibit different acyl CoA specificity, which appears to dictate the type of fatty acid product made. Thus, E. coli makes a mixture of odd and even-number straight chain fatty acids and its KASIII reacts preferentially with acetyl CoA and propionyl CoA (Heath and Rock, 1996. J.Biol.Chem. 271, 10996-11000). Streptomyces glauecescens and Bacillus subtilis make both branched and straight-chain fatty acids. The KASIII from these organisms have been shown to have a more relaxed substrate specificity, reacting with substrates such as isobutyryl CoA as well acetyl CoA and propionyl CoA (Choi, K. H., Heath, R. J. and Rock, C. O. 2000. xcex2-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. J Bacteriol 182: 365-70; Han, L., Lobo, S. and Reynolds, K. A. 1998. Characterization of 3-ketoacyl acyl carrier protein synthase III from Streptomyces glaucescens: Its role in the initiation of fatty acid biosynthesis. J Bacteriol 180: 4481-4486). The KASIII of Mycobacterium tuberculosis on the other hand appears to have a substrate preference for long chain acyl CoA substrates and appears to be responsible for initiation of meromycolate fatty acid biosynthesis (Choi, K. H., Kremer, L., Besra, G. S., and Rock, C. O. 2000. J. Biol. Chem.)
The enzymes involved in type II fatty acid biosynthesis represent attractive targets for modulation of fatty acid synthesis in prokaryotes and plants. Substances that modulate type II fatty acid synthesis could potentially function as therapeutic agents (for example, as antibiotics), or as herbicides. Typically, the screening of potential enzymic effectors is carried out via high throughput screening (HTS) techniques. However, the currently available assay procedures for type II FAS enzymes are inadequate for HTS. For example, the traditional measurement of KASIII activity is a coupled enzyme trichloroacetic acid (TCA) precipitation assay (Han, L. et al. 1998 J.Bacteriol. 180, 4481-4486). In this assay, malonyl CoA-ACP transacylase (FabD, xe2x80x9cMATxe2x80x9d in FIG. 1) is utilized to convert malonyl-CoA to malonyl-ACP. KAS III then catalyzes the condensation of malonyl-ACP with acetyl-CoA which has been radiolabeled, resulting in the production of the radiolabeled product 3-ketoacyl-ACP. An acid precipitation step is then used to physically separate the radioactive 3-ketoacyl-ACP product from the radiolabeled acetyl-CoA substrate. In addition to the precipitation step, an acid-washing step is also required to remove any residual radiolabeled acetyl-CoA from the precipitate. Only after these steps are completed can the radiolabeled, precipitated product be quantitated by scintillation counting.
A variation of this assay method involves binding the 3-ketoacyl ACP product to filter paper disks (such as Whatmann 3MM). These disks are then washed with three changes of ice cold tricchloracteric acid and the filters dried and counted using scintillation cocktail (Choi, Heath and Rock, 2000. J Bacteriol. 182, 365-370). Finally, a gel electrophoresis method has been described. This involves the use of radiolabeled malonyl CoA which is converted in the assay to malonyl ACP by the action of FabD. KASIII then catalyzes the formation of a radiolabeled ketoacyl ACP product using this malonyl ACP and a non-radioactive acyl CoA substrate. The radiolabeled product to reduced to the corresponding 3-hydroxyacyl ACP product by the action of FabG and NADPH and is then resolved from the other components of the assay by use of a polyacrylamide gel. Product quantitation is then obtained by exposure of the gel to a PhosphoImager screen (Choi, Heath and Rock, 2000. J Bacteriol. 182, 365-370).
Obviously, such multistep assay procedures are ill suited for high throughput screening methods. It would be highly desirable to have available a faster and more direct assay for the activities of the enzymes involved in type II fatty acid biosynthesis in order to screen libraries of their potential effectors at an acceptable rate and in a cost-effective manner.
The bacterial fatty acid biosynthesis pathway is a selective target for the development of novel antibiotics for treating infectious disease, (as shown in U.S. Pat. No. 5,614,551 which is incorporated herein by this reference). This selectivity is based both on the significant differences in the fatty acid synthases (FASs) of prokaryotes and eukaryotes and on their relative physiological importance. In higher organisms such as mammals, a multifunctional enzyme complex Type I fatty acid synthase (FAS) in which all of the enzymatic activities are encoded on one or two polypeptide chains, catalyzes the biosynthetic pathway. In humans, this pathway under most conditions is down-regulated due to exogenous dietary lipid intake. In contrast, fatty acid biosynthesis in bacteria appears to be an essential process catalyzed by a set of dissociable enzymes known collectively as a Type II FAS. The natural product thiolactomycin, a thiolactone antibiotic with in vitro and in vivo activity against a number of pathogenic bacteria, selectively inhibits type II, but not type I condensation reactions.
KASIII of Type II FASs has an important regulatory role and catalyzes the first step of fatty acid biosynthesis. This enzyme catalyzes the condensation of malonyl-ACP and acetyl CoA to generate 3-ketoacyl-ACP. Successive elongation steps in the Type II FAS use acyl ACP derivatives as acyl primers (rather than acyl CoA) and are catalyzed by separate 3-ketoacyl-ACP synthases. This pivotal role, the widespread conservation of KASIII among bacteria, and the fact that no mutants totally lacking KASIII activity have been described to date, suggests that this enzyme plays a essential role in bacterial growth. KASIII, therefore represents a promising target for novel antibiotics. Such antibiotics may be effective against bacteria which have become resistant to antibiotics interfering with other essential processes, such as cell-wall biosynthesis. With the exception of thiolactomycin, there are no antibiotics described which specifically target KASIII or other type II ketoacyl synthases. An appropriate assay suitable for high-throughput screening would facilitate the discovery of new KASIII modulators.
A new method of evaluating enzymatic activity in the bacterial type II fatty acid biosynthetic pathway is herein proposed as a solution to the problems outlined above. In a preferred embodiment of the method, in accordance with the present invention, a method to assay enzymatic activity of components of a type II FAS is described in which a reaction mixture is formed by combining a type II fatty acid biosynthetic enzyme, a substrate (ACP or MACP) which has been tagged with a ligand and a radiolabeled acyl or malonyl thioester. The reaction mixture is exposed to a Scintillation Proximity Assay (SPA) support system. The SPA system comprises trapped scintillant and a receptor for the ligand. A level of scintillation which correlates with a level of enzyme activity is measured. The type II fatty acid biosynthetic enzymes that may be assayed by this method include KASI, KASII, KASIII and MAT. The ligand may be biotin and the receptor may be avidin or streptavidin. The thioester may be CoA or ACP, although other thioesters such as those made by N-acetylcysteamine could also be used. The acyl group can vary in much the same way as the acyl thioester specificity of different KAS isozymes does. The APS support system may comprise a bead impregnated with scintillant.
The present invention further provides a method for assessing a compound""s ability to modulate the enzymatic activity of a type II fatty acid biosynthetic enzyme. In the method, a test reaction mixture is formed by combining a type II fatty acid biosynthetic enzyme, a substrate (ACP or MACP) which has been tagged with a ligand and a radiolabeled thioester, and the compound. A control reaction mixture is formed by combining the same components without the compound. Both reaction mixtures are exposed to a Scintillation Proximity Assay (SPA) support system. The SPA system comprises trapped scintillant and a receptor for the ligand. Levels of scintillation which correlates with a level of enzyme activity are measured in both mixtures and the difference between the two levels is determined. The difference is correlated with the ability of the compound to modulate the enzymatic activity of the enzyme. The type II fatty acid biosynthetic enzymes that may be assayed by this method include KASI, KASII, KASIII and MAT. The ligand may be biotin and the receptor may be avidin or streptavidin. The radiolabeled thioester may be malonyl CoA, an acyl CoA, or acyl ACP. The SPA support system may comprise a bead impregnated with scintillant.
The present invention also provides new compositions of matter: biotinylated acyl carrier protein and biotinylated malonyl acyl carrier protein.
The present invention also provides a kit for assaying the enzymatic activity of a type II fatty acid biosynthetic enzyme. The kit includes ACP or MACP which has been tagged with a ligand, a radiolabeled acyl or malonyl thioester, and an SPA support system. Optionally, the kit may also include a type II fatty acid biosynthetic enzyme.
A test sample comprising a target enzyme is contactable with a test compound under suitable conditions that allow the components to interact. A radiolabeled acyl or malonyl thioester substrate and an acyl or malonylacyl carrier protein are added to the components and the enzymatic activity of the target enzyme in the test sample is compared to the enzymatic activity of the target enzyme in a sample not contacted with said test compound, wherein the difference in enzymatic activity in the test sample is indicative of the effect of said test compound on said target enzyme.
In the furtherance of this and other objectives, an assay is provided which is suitable for evaluating enzymatic activity in the bacterial type II fatty acid biosynthetic pathway, said method comprising: (a) contacting a test sample comprising a target enzyme with a test compound under suitable conditions that allow the components to interact; (b) adding to the components a radiolabeled acyl or malonyl thioester substrate and either an acyl carrier protein or malonyl acyl carrier protein; and (c) comparing the enzymatic activity of the target enzyme in the test sample to the enzymatic activity of the target enzyme in a sample not contacted with said test compound, wherein the difference in enzymatic activity in the test sample is indicative of the effect of said test compound on said target enzyme.
A principle object, in accordance with an exemplary embodiment of the present invention, is to provide a screening assay for KAS isozyme inhibitors. In the furtherance of this and other objectives, a rapid assay is provided for measuring the activity of compounds that inhibit KAS from a type II fatty acid synthase comprising, mixing the following components in solution: a test compound, KAS, radiolabeled acyl thioester substrate, a tagged malonyl acyl carrier protein to tagged acyl carrier protein and a product capture element. In particular, a rapid assay is provided for measuring the activity of compounds that affect the activity of xcex2-ketoacyl-ACP Synthase III (KASIII) comprising, mixing the following components in solution: (a) KASIII; (b) the compound, whose effect on KASIII is to be measured; (c) conjugate comprising, a high affinity moiety and an acyl carrier protein; (d) radioactive acyl thioester substrate; and (e) Scintillation Proximity Assay (SPA) support system, where the SPA support system is coated with receptors having high affinity for the ligand moiety of the conjugate; and measuring the radioactivity attached as a result of this specific ligand-receptor interaction.
Yet another objective of the present invention is to provide a faster more direct assay for KASIII activity. In the furtherance of this and other objectives, an assay is provided that does not require either protein precipitation or acid wash steps, or the use of polyacrylamide gels.
An additional objective of the present invention is to provide an assay that is amenable to high throughput screening.
Further objects, features and advantages of the invention will be apparent from the following detailed description taken in conjunction with the accompanying drawings.